First Day Of Culture Research Articles

Ribosomal S6 kinase signaling regulates neuronal viability during development and confers resistance to excitotoxic cell death in mature neurons

The Ribosomal S6 Kinase (RSK) family of serine/threonine kinases function as key downstream effectors of the MAPK signaling cascade. In the nervous system, RSK signaling plays crucial roles in neuronal development and contributes to activity-dependent neuronal plasticity. This study examined the role of RSK signaling in cell viability during neuronal development and in neuroprotection in the mature nervous system. Using neuronal cell-culture-based profiling, we found that suppressing RSK signaling led to significant cell death in developing primary neuronal cultures. To this end, treatment with the RSK inhibitors BiD1870 or SL0101 on the first day of culturing resulted in over 80% cell death. In contrast, more mature cultures showed attenuated cell death upon RSK inhibition. Inhibition of RSK signaling during early neuronal development also disrupted neurite outgrowth and cell growth. In maturing hippocampal explant cultures, treatment with BiD1870 had minimal effects on cell viability, but led to a striking augmentation of NMDA-induced cell death. Finally, we used the endothelin 1 (ET-1) model of ischemia to examine the neuroprotective effects of RSK signaling in the mature hippocampus in vivo. Notably, in the absence of RSK inhibition, the granule cell layer (GCL) was resistant to the effects of ET-1; However, disruption of RSK signaling (via the microinjection of BiD1870) prior to ET-1 injection triggered cell death within the GCL, thus indicating a neuroprotective role for RSK signaling in the mature nervous system. Together these data reveal distinct, developmentally-defined, roles for RSK signaling in the nervous system.

Phytosulfokine alpha enhances regeneration of transformed and untransformed protoplasts of Brassica oleracea.

Phytosulfokine-α (PSK-α) is a disulfated pentapeptide (YIYTQ) acting as an intercellular signal peptide and growth factor. It was originally isolated from conditioned medium of asparagus mesophyll cell cultures in 1996 and later characterized as a hormone-like signal molecule with important roles in numerous processes of in vivo plant growth and development. It is currently becoming a valuable mitogenic factor in plant breeding and biotechnology due to its stimulatory effect on in vitro cell elongation, proliferation and differentiation. The focus of our work was to review current knowledge about the roles of PSK-α in plant biotechnology and to evaluate its influence on the regeneration of protoplasts of four Brassica oleracea cultivars (two cauliflower and two cabbage) cultured under two distinctive protocols and with different protoplast densities. Protoplast regeneration was studied due to its high value for plant genome editing, which is generally limited by the inefficient regeneration of treated protoplasts of numerous important plant genotypes. Our hypothesis was that the stress related to PEG-mediated protoplast transformation and the following decrease in viable protoplast density in culture could be alleviated by the addition of PSK-α to the culture medium. We therefore tested whether PSK-α could increase cell division at the early stages of culture (5 and 15 days after protoplast isolation) and stimulate the formation of microcallus colonies up to the 30st day of culture and to evaluate its influence on callus organogenesis leading to shoot regeneration. The PSK-α showed a strong stimulatory effect on untransformed protoplast regeneration already during the first days of culture, accelerating cell division up to 5.3-fold and the formation of multicellular microcallus colonies up to 37.0-fold. The beneficial influence was retained at later stages of regeneration, when PSK improved shoot organogenesis even if it was present only during the first 10 days of culture. The highest numbers of shoots, however, were regenerated when PSK was present during the first days of culture and later in solid shoot regeneration medium. Finally, the addition of PSK-α to PEG-transformed protoplasts significantly enhanced their division rate and the formation of microcallus colonies in selection media, up to 44.0-fold.

Engineering Anisotropic Cell Models: Development of Collagen Hydrogel Scaffolds with Magneto‐Responsive PEG Microgels for Tissue Engineering Applications

AbstractMimicking tissue‐oriented organization in vitro has been extensively studied in recent years, using both natural and synthetic materials in combination with external magnetic fields to establish anisotropic conditions. Here, a new combination between magneto‐responsive anisometric PEG microgels and collagen hydrogels is explored to establish anisotropic in vitro models. Different sizes of PEG microgels are tested to assess the impact of both width and aspect ratio on the formation and alignment of collagen hydrogels. Results show that the key properties of collagen hydrogels, regarding fibrillogenesis, rheological properties, and fiber diameter are kept consistent upon the combination with PEG microgels. Furthermore, partial collagen fiber alignment is observed when larger (width 10 µm) PEG microgels are employed and magnetically aligned. In vitro studies show cell alignment within the anisotropic collagen hydrogels from the first day in culture. Interestingly, PEG microgels with higher width and length tend to induce less hydrogel contraction even after 7 days in culture. The results demonstrate the ability to establish a 3D unidirectional collagen hydrogel by magnetically aligning anisometric microgels during the gelation process, which can be promising for different tissue engineering applications.

Exploring the effects of temperature and light availability on the vegetative propagation processes of the non-native species Asparagopsis armata

Widespread generalist species, particularly the non-native invasive ones, are expected to be enhanced by climate change resulting in a biotic homogenization of ecosystems. The red seaweed Asparagopsis armata is a non-native opportunistic species, widely distributed in the European coasts of the North Atlantic, where it has been considered invasive. In this work, we examined the effects of temperature and irradiance on the vegetative propagation process of this species in a laboratory experiment. We considered vegetative propagation due to its implications in the invasion process (as it is considered one of the main sources of recruitment). In gametophytes, the process was characterized through the survival rates of hook-shaped specialized structures and the production and growth of new plantlets from hooks of 1-3cm in length. In tetrasporophytes, the growth and phycobiliprotein contents of previously excised tufts was analyzed. For gametophytes, results revealed how vegetative propagation for this species was conditioned by the low survival rates of hooks once detached from the main thallus. In our experiment, survival probabilities after 30 days of culture were always below 50%. Comparisons among environmental conditions showed lower survival rates under increasing levels of temperature and decreasing levels of irradiance. In fact, mass mortality was detected at 18°C and low irradiance, where almost any hook-shaped fragment survived. Nevertheless, patterns of appearance and growth of plantlets at different temperatures and irradiances were not clear. In the case of tetrasporophytes, only positive growth rates were registered at 15°C and 55-60 µmol/m2/s after 30 days of culture. Higher concentration of phycobiliproteins was detected at higher temperatures during the first days of culture, while not clear patterns were detected at the end. In the light of climate change, understanding these reproduction patterns is necessary in order to adopt better management actions in the future.

Sarcomeric network analysis of ex vivo cultivated human atrial appendage tissue using super-resolution microscopy

Investigating native human cardiac tissue with preserved 3D macro- and microarchitecture is fundamental for clinical and basic research. Unfortunately, the low accessibility of the human myocardium continues to limit scientific progress. To overcome this issue, utilizing atrial appendages of the human heart may become highly beneficial. Atrial appendages are often removed during open-heart surgery and can be preserved ex vivo as living tissue with varying durability depending on the culture method. In this study, we prepared living thin myocardial slices from left atrial appendages that were cultured using an air-liquid interface system for overall 10 days. Metabolic activity of the cultured slices was assessed using a conventional methyl thiazolyl tetrazolium (MTT) assay. To monitor the structural integrity of cardiomyocytes within the tissue, we implemented our recently described super-resolution microscopy approach that allows both qualitative and quantitative in-depth evaluation of sarcomere network based on parameters such as overall sarcomere content, filament size and orientation. Additionally, expression of mRNAs coding for key structural and functional proteins was analyzed by real-time reverse transcription polymerase chain reaction (qRT-PCR). Our findings demonstrate highly significant disassembly of contractile apparatus represented by degradation of alpha -actinin filaments detected after three days in culture, while metabolic activity was constantly rising and remained high for up to seven days. However, gene expression of crucial cardiac markers strongly decreased after the first day in culture indicating an early destructive response to ex vivo conditions. Therefore, we suggest static cultivation of living myocardial slices derived from left atrial appendage and prepared according to our protocol only for short-termed experiments (e.g. medicinal drug testing), while introduction of electro-mechanical stimulation protocols may offer the possibility for long-term integrity of such constructs.

P-134 An artificial intelligence method based on time-lapse images and deep learning may predict if a day2/3 embryo will form a utilizable blastocyst

Abstract Study question Can iDAScore v2.0 predict likelihood of development into a utilizable blastocyst from time-lapse images captured during the first two or three days of embryo development? Summary answer iDAScore v2.0 predicts utilizable blastocyst formation from time-lapse images of early embryo development. Specific score thresholds can be established to ensure high specificity of prediction. What is known already Treatment efficacy would benefit from a reliable method to predict the potential of early embryos to develop into utilizable blastocysts and select fresh IVF treatment cycles suitable for blastocyst culture. Reports indicate that time-lapse images captured during the first few days of development and analyzed by deep learning methods may be an accurate approach. A fully automated AI model, iDAScore, is reported to be more accurate than routine morphology and morphokinetics for prediction of implantation. However, the applicability of iDAScore calculated on day 2 and 3 as a tool to predict blastocyst development has yet to be tested. Study design, size, duration This is a retrospective multicenter cohort study including time-lapse videos of 9111 embryos cultured to blastocyst stage, from 1283 fresh IVF treatments between 2016-2021 at two ART clinics together performing > 2000 OPU per year. Images captured during the first days of culture were used to calculate iDAScores (at 48 and 66 hours post insemination) and determine the ability to discriminate embryos likely to develop into utilizable blastocysts according to Gardner score of 3BB or more. Participants/materials, setting, methods Patients were required to be undergoing IVF treatment using their own (autologous) oocytes whereby all zygotes were cultured up to the blastocyst stage in GTL® culture medium using the Embryoscope+® time-lapse system (Vitrolife, Gothenburg, Sweden). At least two experienced embryologists graded blastocysts on day 5/6, selecting utilizable blastocysts for transfer/vitrification. To evaluate the performance of iDAScore to predict development into utilizable blastocysts, sensitivity, specificity, and area under ROC curve (AUC) values were calculated. Main results and the role of chance The blastocyst utilization rate was 43.8% (3990 utilizable blastocysts of 9111 day 2/3 embryos developing from 2PN zygotes). Prediction of utilizable blastocysts obtained AUCs of 0.768 (0.759 - 0.778) and 0.782 (0.773 - 0.791) for day 2 and day 3, respectively. These results suggest IDAScore prediction performed slightly better when calculated on day 3 than on day 2. The ability to discriminate between utilizable and non-utilizable blastocysts at different thresholds of iDAScore on day 2 and on day 3, suggests that embryos should have a minimum iDAscore of 5 on day 2 to obtain a high specificity of 89%, and a threshold of 6 on day 3 to obtain a high specificity of 88%. Lower sensitivities were observed for these potential thresholds, 35% on day 2 and 41% on day 3. Limitations, reasons for caution This is a retrospective analysis where most cycles have at least five zygotes that supported subsequent blastocyst development, limiting relevance for patients with fewer zygotes. IDAScore is developed to predict implantation potential, not utilizable blastocyst, thus could be an underestimation of the potential performance of a model for utilization prediction. Wider implications of the findings The iDAScore v2.0 may provide valuable information for embryologists and be useful as a prognostic tool to select patients with high chance to obtain blastocysts based on day2/3 time-lapse images and thereby may achieve similar live birth rate per transfer with no need for extended culture. Trial registration number Not applicable

JAK inhibitors differentially modulate B cell activation, maturation and function: A comparative analysis of five JAK inhibitors in an in-vitro B cell differentiation model and in patients withrheumatoid arthritis.

Janus kinase (JAK) inhibitors have been approved for the treatment of several immune-mediated diseases (IMIDs) including rheumatoid arthritis (RA) and psoriatic arthritis and are in clinical trials for numerous other IMIDs. However, detailed studies investigating the effects of different JAK inhibitors on B cells are missing. Within this study, we therefore aimed to characterize the effect of JAK inhibition on the B cell compartment. To this end, we investigated the B cell compartment under JAK inhibition and compared the specific effects of the different JAK inhibitors tofacitinib (pan-JAK), baricitinib (JAK1/2), ruxolitinib (JAK1/2), upadacitinib (JAK1/2) as well as filgotinib (selective JAK1) on in-vitro B cell activation, proliferation, and class switch recombination and involved pathways. While B cell phenotyping of RA patients showed an increase in marginal zone (MZ) B cells under JAK inhibition, comparison with healthy donors revealed that the relative frequency of MZ B cells was still lower compared to healthy controls. In an in-vitro model of T-cell-independent B cell activation we observed that JAK1/2 and selective JAK1 inhibitor treatment led to a dose-dependent decrease of total B cell numbers. We detected an altered B cell differentiation with a significant increase in MZ-like B cells and an increase in plasmablast differentiation in the first days of culture, most pronounced with the pan-JAK inhibitor tofacitinib, although there was no increase in immunoglobulin secretion in-vitro. Notably, we further observed a profound reduction of switched memory B cell formation, especially with JAK1/2 inhibition. JAK inhibitor treatment led to a dose-dependent reduction of STAT3 expression and phosphorylation as well as STAT3 target gene expression and modulated the secretion of pro- and anti-inflammatory cytokines by B cells. JAK inhibition has a major effect on B cell activation and differentiation, with differential outcomes between JAK inhibitors hinting towards distinct and unique effects on B cell homeostasis.

Bioactive Lactose-Modified Chitosan Acts as a Temporary Extracellular Matrix for the Formation of Chondro-Aggregates

Spheroids based on chondrocytes are gaining interest in the treatment of injured cartilage tissue due to their regenerative potential. In this study, chondrocyte-based aggregates were formed by culturing cells on a polymeric coating of a lactose-modified chitosan (CTL) and their evolution was followed for 14 days of culture. Morphological analyses through scanning electron microscopy indicated that these cell aggregates formed by a process of cell aggregation rather than proliferation, resulting in structures with an irregular morphology that were up to 1 mm in size. Chondrocytes interact very closely with CTL. In the early days of cellular aggregation, CTL is distributed among the cells, while in later stages, it is localized in the inner part of the aggregates, forming an amorphous matrix. The initial outer position of the polymer is replaced over time by an increased matrix deposition whose main component is type II collagen. Transmission electron microscopy pointed out that the chondrocytes internalized the polymer during the first days of culture. The presence of CTL does not affect the ability of cells to migrate from chondro-aggregates when transferred to uncoated wells, mimicking the ability of cells to colonize a cartilage defect. Overall, these results suggest that CTL plays a role as a temporary matrix for chondrocytes aggregation during chondro-aggregates formation. This opens up innovative therapeutic approaches for cartilage regeneration without the need to use a scaffold to deliver cells to the damaged site.

Viability of mitochondria-labeled retinal ganglion cells in organotypic retinal explant cultures by two methods

Retinal explant cultures provide a valuable system to study retinal function in vitro. This study established a new retinal explant culture method to prolong the survival of retinal ganglion cells (RGCs). Explants were prepared in two different ways: with or without optic nerve. Retinas from newborn mice that had received an injection of MitoTracker Red into the contralateral superior colliculus to label axonal mitochondria were cultured as organotypic culture for 7 days in vitro. At several time points during the culture, viability of RGCs was assessed by multi-electrode array recording, and morphology by immunohistochemical methods. During the culture, the thickness of the retinal tissue in both groups gradually decreased, however, the structure of the layers of the retina could be identified. Massive apoptosis in the retinal ganglion cell layer (GCL) appeared on the first day of culture, thereafter the number of apoptotic cells decreased. Glial activation was observed throughout the culture, and there was no difference in morphology between the two groups. RGCs loss was exacerbated on 3rdday of culture, and RGCs loss in retinal explants with preserved optic nerve was significantly lower than in retinas that did not preserve the optic nerve. More and longer-lasting mitochondrial signals were observed in the injured area of the optic nerve-preserving explants. Retinal explants provide an invaluable tool for studying retinal function and developing treatments for ocular diseases. The optic nerve-preserving culture helps preserve the integrity of RGCs. The higher number of mitochondria in the nerve-preserving cultures may help maintain viability of RGCs.

Spent media analysis suggests cultivated meat media will require species and cell type optimization

Cell culture media design is perhaps the most significant hurdle currently facing the commercialization of cultivated meat as an alternative source of dietary protein. Since media optimization for a specific culture system requires a significant amount of effort and investment, a major question remaining is whether media formulations can be easily shared across multiple production schemes for cells of different species and lineages. Here, we perform spent medium analysis to compare the specific nutrient utilization of primary embryonic chicken muscle precursor cells and fibroblasts to the murine C2C12 myoblast cell line. We demonstrate that these related cell types have significantly different nutrient utilization patterns collectively and on a per-cell basis, and that many components of conventional media do not appear to be depleted by the cells. Namely, glucose was not consumed as rapidly nor as completely by the chicken muscle precursors compared to other cells overall, and there were significant differences in specific consumption rates for several other key nutrients over the first day of culture. Ultimately, our results indicate that no one medium is likely ideal and cost effective to culture multiple cell types and that novel methods to streamline media optimization efforts will be important for the industry to develop.

RANKL interferes with osteoclastogenesis in PEG-fused U937 cells through LGR4

ABSTRACT Introduction RANKL plays an important role in the differentiation and maturation process of preosteoclast cells. The osteoclast is a multinucleated cell that can have various sizes and a variable number of nuclei. However, there are no models that allow us to understand how successive cell fusions have a limit, or how cell fusion is regulated. Methodology The present investigation was aimed to determine whether fusing U937 cells with PEG to generate osteoclast-like cells expresses LGR4 and whether applying RANKL to these cells modifies osteoclastic activity compared to non-PEG-fused and RANKL-treated cells. Results By fusing U937 cells with PEG, it was found that the LGR4 receptor expression was promoted as early as 24 hours of culture. Applying RANKL before or after fusion inhibits osteoclastic activity. Interfering RANKL interaction with LGR4 in PEG-treated cells recovers and increases cell fusion and osteoclastic activity. PEG-fused U937 cells show osteoclast markers similar to those observed in the classical RANKL-stimulated cell model. Conclusion Our model allows us to understand that RANKL has fusogenic activity during the first days of culture and in fused cells modulates fusion, contributing to differentiate the role of RANKL before and after fusion through LGR4.

Determination of Antibiotic Susceptibility of Bacteria Isolated From Endotracheal Tube in Pulmonary Intensive Care Unit of a Hospital in Bandar Abbas

Background: The present study attempted to determine the antibiotic susceptibility of bacteria isolated from endotracheal tube culture on patients admitted to the intensive care units (ICUs) of Shahid Mohammadi hospital in Bandar Abbas. Materials and Methods: This cross-sectional study was conducted on 114 patients hospitalized in the pulmonary ICU of Shahid Mohammadi hospital in Bandar Abbas from March 2018 to March 2019. The samples of the patients’ endotracheal tube secretions were collected for the culture purpose on the first and fifth days of hospitalization in the ICU where the disk diffusion method was used to determine antibiotic susceptibility. Results: The most common isolated microorganisms from the first day of culture were 26 Acinetobacter spp. isolates (22.8%), 10 Candida isolates (8.8%), and 9 Pseudomonas aeruginosa isolates (7.9%). The most common microorganisms which were isolated on the fifth day comprised Acinetobacter spp. with 57 isolates (51.8%), Candida with 6 isolates (5.5%), and P. aeruginosa with 6 isolates (5.5%). The highest resistant bacteria isolated from Acinetobacter spp. samples on the first-day were amikacin (92.3%) and meropenem (84.6%). The highest resistance of P. aeruginosa was against meropenem (77.8%), while the highest resistance of Acinetobacter spp. isolates was found to be amikacin (94.7%) and meropenem (86%) from the fifth-day samples. Further, P. aeruginosa isolates had the highest resistance in meropenem (83.3%), co-trimoxazole, and cefepime (66.7% for each of them). Conclusion: Ventilator-associated pneumonia (VAP) is considered a warning condition due to multidrug-resistant bacteria. According to the present study, the most common VAP-causing bacteria are gram-negative, especially Acinetobacter, with high antibiotic resistance.

Embryonic Development of Avian Pineal Secretory Activity-A Lesson from the Goose Pineal Organs in Superfusion Culture.

The embryonic ontogeny of pineal secretory activity in birds has been investigated almost exclusively in chickens. This study aimed to characterize this process in domestic geese. The pineal organs of embryos aged 18–28 days were incubated in superfusion culture under different light conditions for 4–5 days and treated with norepinephrine (NE). Melatonin (MLT) was measured by radioimmunoassay and other indoles by HPLC with fluorescence detection. Additionally, pineal organs were collected from embryos at 14–28 days of age and used to measure catecholamines by HPLC with electrochemical detection. MLT secretion increased with embryo age, most intensively between the 22nd and 24th days of life. The daily changes in MLT secretion under the 12 L:12D cycle occurred on the first day of culture, starting from an embryonic age of 24 days. MLT secretion was controlled by the light-dark cycle in all age groups studied. However, exposure to light during the scotophase did not alter the secretion of MLT. The endogenous oscillator expressed its activity in regulating MLT secretion in the pineal organs of embryos aged 24 days and older but could not generate a rhythm after one cycle. The rhythm of 5-hydroxytryptophan release during the first day of culture was found in the pineal organs of all embryos, while the rhythmic release of N-acetylserotonin and 5-methoxyindole acetic acid started at the age of 24 days. The proportion of released indoles changed with embryo age. NE caused a decrease in MLT secretion and provoked an increase in serotonin release. Incubation of the pineal organs induced the development of MLT secretory machinery and its diurnal rhythmicity. The pineal content of catecholamines increased prominently at the end of embryonic development.

Phospholipase A activity and the biocontrol potential of Trichoderma harzianum and Trichoderma atroviride

ABSTRACT The aim of this work was to study the phospholipase A (PLA) enzyme activity of extracts from Trichoderma harzianum 1A and Trichoderma atroviride αCp8 culture media. Fermentation on submerged culture was performed and PLA activity of extracts was evaluated as a function of culture time. A turbidimetric method for continuous analysis of phospholipase activity was applied. By this, vesicles rather than soluble substrates are used resembling the in vivo environment. In Trichoderma’s culture experiments, lecithin phospholipids as inductors promoted a PLA expression level of the same order to the corresponding strain control assay. T. harzianum developed the highest PLA activity and was considered as the most potent strain. Furthermore, the activity level was clearly greater when the culture medium included the plant pathogenic fungus Sclerotium rolfsii. The T. harzianum strain showed the highest PLA potential. Besides, both T. harzianum and T. atroviride, developed higher PLA activity at the first days of culture. In the present work, T. harzianum 1A and T. atroviride αCp8 at a lower level, showed an important PLA potential for S. rolfsii control. On the other side, a lower relative response was observed when both Trichoderma strains were assayed against other pathogens such as Macrophomina phaseolina and Rhizoctonia solani. The present work contributes to the scientific knowledge about PLA activity in Trichoderma and could be useful for technological purposes to prepare formulations for the biological control of S. rolfsii in agriculture.

Clinical Microbiology Perspective of Empyema caused by Streptococcus constellatus in Malignancy Patient: A Case Report

Introduction: Streptococcus constellatus (SC) is commensal bacteria and belongs to Streptococcus anginosus group (SAG). However, SC causes infections especially in patient with underlying diseases. SC empyema is a clinical case that is described in very few studies, especially in Indonesia.Case: A 45-year-old man was admitted to emergency department in Dr. Soetomo General Hospital Surabaya with respiratory insufficiency on November 11th 2020 after 3 months of non-productive cough and a week of weakness. He was diagnosed with empyema on right hemithorax and received chest tube insertion. SC was isolated from pleural fluid sample after first day of culture on Bactec BD bottle. GeneXpert result of pleural fluid was negative for Mycobacterium tuberculosis (MTB). He completed 10 days of intravenous ampicillin-sulbactam and metronidazole. The CT scan reported solid mass of 4.7x7.4x7.8 cm in posterolateral segment of inferior lobe right lung, right pleural effusion, with adenocarcinoma as biopsy result.Discussion: SC is a normal commensal in respiratory tract, however with the presence of a certain factor such as immunocompromised, colonized SAG directly induces an infection after entering normal sterile sites in the body including pleural fluid.Conclusion: Although infection caused by SC is a rare case, it still should be considered in clinical diagnosis and treatment of related infections, particularly in patients with comorbidities. The prognosis was good with appropriate antibiotics and chest tube insertion.

Mortality rate of newly hatched larvae of Nile tilapia (Oreochromis niloticus) submitted to different photoperiods

ABSTRACT: Fish farming is a growing area in the aquaculture industry. Brazil is among the five largest tilapia producers in the world, with a production of 283 thousand tons in 2017. Tilapia has been intensively cultivated and improved through more efficient cultivation techniques. In this sense, the objective was to evaluate the mortality rate of newly hatched larvae of Nile tilapia reared three photoperiods (12Light:12Dark, 18L:06D, and 24L:0D) during 9 days of cultivation. For this purpose, a recirculation system with 9 water tanks was set up, with 3 boxes per group containing 930L of freshwater. After the embryos hatched, the larvae were transferred to nurseries (2 trays per tank, n = 64 larvae/tray), with 384 larvae per photoperiod. Water quality parameters were monitored and remained in the ideal ranges between treatments. However, temperature (p=0.004) and pH values (p= 0.009) for the 18L:06D photoperiod were lower than those observed for the 24L:0D photoperiod. A high mortality rate was observed in the first days of culture, specifically on the second day, for all photoperiods. However, there was a decrease in mortality throughout the experiment, with little variation in the mortality rate for the remainder of the experiment. In this context, it appears that the influence of the photoperiod on the survival of the fish seems to be related to the age of the animal. KEYWORDS: Tilapia farming, Survival, Light stimulation.

Prolonged cultured human hepatocytes as an in vitro experimental system for the evaluation of potency and duration of activity of RNA therapeutics: Demonstration of prolonged duration of gene silencing effects of a GalNAc-conjugated human hypoxanthine phosphoribosyl transferase (HPRT1) siRNA

We report here the evaluation of a novel in vitro experimental model, prolonged cultured human hepatocytes (PCHC), as an experimental system to evaluate the potency and duration of effects of oligonucleotide therapeutics. A novel observation was made on the redifferentiation of PCHC upon prolonged culturing based on mRNA profiling of characteristic hepatic differentiation marker genes albumin, transferrin, and transthyretin. Consistent with the known de-differentiation of cultured human hepatocytes, decreases in marker gene expression were observed upon culturing of the hepatocytes for 2 days. A novel observation of re-differentiation was observed on day 7 as demonstrated by an increase in expression of the marker genes to levels similar to that observed on the first day of culture. The expression of the differentiation marker genes was highest on day 7, followed by a gradual decrease but remained higher than that on day 2 for up to the longest culture duration evaluated of 41 days. The redifferentiation phenomenon suggests that PCHC may be useful for the evaluation of the duration of effects of oligonucleotide therapeutics on gene expression in human hepatocytes. A proof of concept study was thereby conducted with PCHC with a GalNAc-conjugated siRNA targeting human hypoxanthine phosphoribosyl transferase1 (HPRT1). HPRT1 mRNA expression in siRNA-treated cultures decreased to 21% of that in untreated hepatocytes on day 1, <10% from days 2 to 12, <20% from days 16 to 33, and eventually recovered to 64% by day 41. Our results suggest that PCHC represent a clinically-relevant cost- and time-efficient experimental tool to aid in the evaluation of GalNAc-siRNA silencing activity, providing information on both efficacy and duration of efficacy. PCHC may be applicable in the drug development setting as a species- and cell type-relevant experimental tool to aid the development of oligonucleotide therapeutics.

Retrovirus-mediated gene transfer in polyclonal T cells results in lower apoptosis and enhanced ex vivo cell expansion of CMV-reactive CD8 T cells as compared with EBV-reactive CD8 T cells

AbstractTo modulate alloreactivity after hematopoietic stem cell transplantation, “suicide” gene-modified donor T cells (GMCs) have been administered with an allogeneic T-cell–depleted marrow graft. We previously demonstrated that such GMCs, generated after CD3 activation, retrovirusmediated transduction, and G418 selection, had an impaired Epstein-Barr virus (EBV) reactivity, likely to result in an altered control of EBV-induced lymphoproliferative disease. To further characterize the antiviral potential of GMCs, we compared the frequencies of cytomegalovirus (CMV)–specific CD8+T (CMV-T) cells and EBV-specific CD8+T (EBV-T) cells within GMCs from CMV- and EBV-double seropositive donors. Unlike anti-EBV responses, the anti-CMV responses were not altered by GMC preparation. During the first days of culture, CMV-T cells exhibited a lower level of CD3-induced apoptosis than did EBV-T cells. In addition, the CMV-T cells escaping initial apoptosis subsequently underwent a higher expansion rate than EBV-T cells. The differential early sensitivity to apoptosis could be in relation to the “recent activation” phenotype of EBV-T cells as evidenced by a higher level of CD69 expression. Furthermore, EBV-T cells were found to have a CD45RA–CD27+CCR7–effector memory phenotype, whereas CMV-T cells had a CD45RA+CD27–CCR7–terminal effector phenotype. Such differences could be contributive, because bulk CD8+CD27–cells had a higher expansion than did bulk CD8+CD27+cells. Overall, ex vivo T-cell culture differentially affects apoptosis, long-term proliferation, and overall survival of CMV-T and EBV-T cells. Such functional differences need to be taken into account when designing cell and/or gene therapy protocols involving ex vivo T-cell manipulation.

Upregulation of PI3K/AKT/PTEN pathway is correlated with glucose and glutamine metabolic dysfunction during tamoxifen resistance development in MCF-7 cells

Tamoxifen resistance is emerging as a big challenge in endocrine therapy of luminal A breast cancer patients. In this study, we aimed to determine the molecular changes of PI3K/AKT/PTEN signaling pathway during tamoxifen-resistance development using gradually increased doses of tamoxifen in one model, while fixing tamoxifen treatment dose at 35 μM for several times in the second model. An upregulation of AKT/PI3K genes was noticed at 30 μM tamoxifen concentration in cells treated with a gradual increase of tamoxifen doses. In the second model, significant upregulation of AKT1 was seen in cells treated with 35 μM tamoxifen for three times. All genes studied showed a significant increase in expression in resistant cells treated with 50 µM and 35 µM six times tamoxifen. These genes’ upregulation was accompanied by PTEN and GSK3 ß genes’ down-regulation, and it was in correlation to the changes in the metabolic rate of glucose in tamoxifen-resistant models. A significant increase in glucose consumption rate from culture media was observed in tamoxifen resistant cells with the highest consumption rate reported in the first day of culturing. Increased glucose consumption rates were also correlated with GLUL significant gene expression and non-significant change in c-MYC gene expression that may lead to increased endogenous glutamine synthesis. As a result, several molecular and metabolic changes precede acquired tamoxifen resistance could be used as resistance biomarkers or targets to reverse tamoxifen resistance.

A high glucose concentration during early stages of in vitro equine embryo development alters expression of genes involved in glucose metabolism.

Equineembryos exhibit an unusual pattern of glucose tolerance in vitro and are currently cultured in hyperglycaemic conditions. Our main objective was to analyse the effect of different glucose concentrations on in vitro-produced equine embryo development and quality. Experiments comparing in vitro and in vivo produced embryos. Oocytes (n=641) were collected from post-mortem ovaries, matured in vitro and fertilised by intracytoplasmic sperm injection (ICSI). Embryo culture was divided from Day 0 to Day 4 and from Day 4 to Day 9 in three groups: 5-10 (5 and 10mmol/L glucose respectively; n=87); 5-17 (5 and 17.5mmol/L; n=66); and 10-17 (10 and 17.5mmol/L; n=117). A control group of 20 in vivo produced blastocysts was included. Cleavage and blastocyst rates were evaluated and embryos were snap-frozen for analysis of the relative mRNA expression of genes related to mitochondrial function, DNA methylation, apoptosis, glucose transport and metabolism. No differences were observed in the cleavage or blastocyst rates among in vitro groups. Under high glucose conditions in vitro (10-17 group), BAX/BCL2 was higher, and PFKP, LDHA and COX2 were overexpressed compared to all other groups. The two groups with 5mmol/L glucose concentration during the first culture stage (5-10 and 5-17) displayed similar patterns which differed to the 10-17 group. Conclusions related to embryo quality are based on gene expression patterns. Transfer of in vitro-produced embryos would reveal whether the observed differences improve embryo developmental competence. Five mM glucose during the first days of culture seems to be preferable to avoid over-activation of embryonic glycolytic pathways. Further studies are necessary to determine whether this improves embryo developmental competence.